Crosstalk of Splicing and Signaling during HSC Formation Project Abstract Cell fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of cell fate choice is well-studied, the regulatory role of RNA splicing in this process is poorly understood. Indeed, as RNA splicing is mostly studied in yeast and cell culture, tissue-specific regulation of splicing in vivo is largely unexplored. Here, using hematopoietic stem cell (HSC) formation as a paradigm for cell fate specification, we seek to illuminate how the molecular regulation of RNA splicing impacts cell fate. During embryogenesis, a subset of blood-forming (i.e. `hemogenic') endothelial cells undergoes a major cell fate transition from an `endothelial' identity to a nascent HSC. Understanding this transition is critical for advancing both HSC-based regenerative medicine and our basic understanding of blood genesis. We recently reported a severe HSC formation defect in zebrafish loss- of-function mutants for the spliceosomal component, splicing factor 3b, subunit 1 (sf3b1). Surprisingly, we found that this presumed constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. Our central premise is that splice isoform selection is a highly coordinated process critical for HSC formation. We hypothesize that the splicing factor Sf3b1 is central to this coordination via directing splice isoform choice of signaling pathway components essential for HSC formation, and that in turn these signaling pathways regulate Sf3b1-mediated splicing outcomes. In this proposal, we will use zebrafish genetic and screening capabilities to address two aims: (1) to determine how splicing impacts HSC fate choices by exploring the role of specific splice isoforms on HSC formation and defining the function of cis-regulatory elements on Sf3b1-regulated splicing choices, and (2) to determine signaling pathways impacting HSC splicing outcomes using chemical screening and genomics approaches. Accomplishing these aims will inform our fundamental understanding of the poorly understood role of splicing in hematopoietic lineage fate choice.